Summary Integrins are the major cell adhesion molecules used by virtually all cells to migrate. During adhesion, integrins are clustered to form a variety of complexes including nascent adhesions, small focal complexes and mature focal adhesions. Through these sites, the cell exerts tension on the substratum. Much of what we know about the assembly of adhesion complexes comes from studies on cells detached and replated on extracellular matrix (ECM) ligands. This cell spreading assay has been powerful for understand mechanisms of adhesion formation, yet it does not fully recapitulate key features and behaviors of adhesion formation during cell migration. In particular, it does not account for the polarized formation of adhesions near the front of the cell and does not take into account the endocytic recycling of integrins, which is critical for cell migration in both 2D and 3D environments. We have developed a new focal adhesion assembly assay that is critically dependent on recycled integrin derived from the endocytosis of integrins in adhesions. Interestingly, this integrin travels in an unliganded and active conformation and leads to the highly polarized formation of adhesions at the leading edge of migrating cells. We will use cell biological, biochemical and high resolution imaging approaches to test the hypothesis that recycled integrin acts to seed new adhesion formation near sites of its exocytosis near the leading edge. Our three aims are: 1) to examine the relationship between integrin recycling, activation state, exocytic sites and adhesion formation, 2) to determine how the microtubule cytoskeleton contributes to the polarized formation of adhesions from recycled integrins and, 3) to determine how extracellular matrix topology and stiffness affect adhesion formation from recycled integrins. Throughout, we will examine how the formation of adhesions from recycled integrins affect the ability of cells to migrate. This work will advance understanding of the basic mechanisms cells use to polarize their adhesions for cell migration, which may lead to new strategies for intervention in cases where cell migration is unregulated, such as cancer metastasis and persistent inflammation.